ETS (E-Twenty-Six) transcription factors are found throughout the body and play a role in a variety of different physiological functions, including cell differentiation, cell proliferation, apoptosis, angiogenesis, cell migration, and cell cycle control. They are implicated in a wide variety of pathophysiologies, including cancer. One member of the ETS family is designated PU.1, which regulates the expression of receptors such as IL-2Rγ, IL-7Rα and Toll-like receptors. As such, PU.1 is also involved in various autoimmune diseases. Because sequence specific binding is a necessary step in ETS-mediated gene activation, inhibition of the ETS-DNA complex can serve as the pharmacological basis for the treatment of a wide variety of diseases.
Acute myeloid leukemia (AML) is a cancer of the hematopoietic system, characterized by the abnormal clonal proliferation of immature cells, following various genetic and epigenetic alterations. Despite efforts to discover novel therapeutic options, survival in AML remains poor, with a 5-year overall survival of 25%, with overall outcome being worst for patients >60 years of age who represent the vast majority. Especially in this age group, clinical outcome has not significantly improved in the past 4 decades. AML is a genetically very heterogeneous disease, characterized by recurrent genetic mutations which often occur in combination in individual patients (about 30 mutations recur in patients at a frequency of >1%), and on average patients with AML carry a combination of 3-5 ‘driver mutations’. One of the major challenges facing currently ongoing ‘precision oncology’ efforts is the low frequency of a larger number of individual mutations and their combinatorial occurrence. Instead of targeting specific genetic aberrations, an alternate strategy for AML treatment would be targeting of more commonly dysregulated pathways that are implicated in various AML subtypes and in larger subsets of patients.
Over the last 15 years, increasing evidence has shown the critical importance of PU.1, an ETS family transcription factor, in AML. A functionally critical decrease in PU.1 level has been described in FLT3-ITD, RUNX1-ETO and promyelocytic leukemia, representing 24, 7 and 13% of all AMLs, respectively (cancer.sanger.ac.uk). Additionally, PU.1 loss of function heterozygous mutations or deletions have been described in AML, and are found in ˜10% of MLL-translocated AML. Homozygosity of a single nucleotide variant in an upstream regulatory element (URE) of PU.1, lowering PU.1 expression, has been described in AML with complex karyotype, and a study on highly purified stem cells of patients with AML showed reduced PU.1 levels in at least 40% of examined cases. Overall, disruption of PU.1 expression or activity is present in more than half of AML patients and is associated with a specific transcriptional and epigenetic program, rendering it a very attractive potential therapeutic target.
PU.1 is highly conserved between humans and mice and its functions have been studied using a number of genetically engineered mouse models, which have further proven PU.1's crucial role in hematopoiesis. PU.1 is essential for myeloid and lymphoid lineages, as well as hematopoietic stem cell (HSC) maintenance. Its role in AML development has been firmly established through mouse models with reduced, but not completely absent, PU.1 expression. Homozygous knockout of an enhancer (URE) located −14 kb upstream of PU.1 leads to a decrease in PU.1 expression of 80% and development of a stem cell-derived AML between 3 to 8 months of age. Enhancer haplodeficiency of PU.1 is not sufficient to induce leukemia by itself; however it leads to myeloid bias in stem cells and AML development in combination with cooperating events.
Thus, PU.1 and its downstream transcriptional network are crucial in hematopoiesis and leukemogenesis. AML with disruption of PU.1 function is a distinct entity, associated with specific oncogenes, as well as specific molecular signatures. Thus, targeting PU.1 in AML could be an appealing option for treatment. In the past, strategies to rescue PU.1 expression in AML cells have been explored. Overexpression of PU.1 is sufficient to trigger neutrophil differentiation in acute promyelocytic leukemia (APL), and leads to differentiation and apoptosis of various primary AML samples. Unfortunately, elevation of PU.1 levels or activity is difficult to achieve pharmacologically. However, as complete loss of PU.1 leads to stem cell failure, AML cells may be more vulnerable to further PU.1 inhibition in comparison to normal hematopoietic cells.
In addition to hematologic malignancies, PU.1 is also a promising target in a range of non-malignant diseases with an immunological basis, in which pharmacological inhibition represents a novel therapeutic strategy. The essential role of PU.1 in the differentiation and development of myeloid lineages is well established in mouse and human models of hematopoiesis. PU.1 induces the expression of key receptors such as TLR4 and GM-CSFR, which sensitize granulocytes and monocytes to endotoxins and specific pro-inflammatory cytokines. Thus, PU.1 represents an attractive therapeutic target in non-malignant inflammatory diseases in which granulocytes and monocytes are major cellular mediators. Examples of such diseases that are mediated, at least in part, by granulocytes/monocytes include (but are not restricted to) endotoxemia, rheumatoid arthritis and neurodegenerative diseases.
In mouse models of peritonitis, GM-CSF stimulates differentiation of tissue macrophages and sensitization to bacterial endotoxins (LPS) in a PU.1-dependent manner that strongly correlates with mortality and is markedly attenuated in GMCSF-deficient animals. In other mouse models, endotoxins potently stimulates TLR4 on mature macrophages, leading to local (e.g., lung) and systemic inflammation that is blunted in PU.1-deficient chimeric animals.
The widespread clinical use of anti-TNFα antibodies has highlighted the central role of macrophages/monocytes in RA. Up-regulation of PU.1 is a common feature in activated synovial macrophages and is associated with TLR4 expression. Attention to PU.1 as a therapeutic target in RA is also increasing owing to its regulation of microRNA expression, especially miR-155, a pro-inflammatory RA regulator analogous to its role in AML. A recent observational study has also found elevated PU.1 expression in patients with systemic lupus erythematosus.
Recent evidence has brought to light the immunological basis of chronic neurodegenerative diseases, including Alzheimer's disease. Specifically, microglial proliferation, mediated via the PU.1-target gene csf1r, is associated with neuronal damage and disease progression in mouse models of chronic neurodegeneration. A broader involvement of PU.1 transactivation in microglial development has been identified, and most recently in the specific case of Alzheimer's disease.
In addition to its importance as a myelopoietic regulator, PU.1 also plays an essential role in the function and polarization of certain mature T helper cells. Secretion of IL-9 by Th9 cells, a major cytokine of in allergic inflammation, is transcriptionally controlled by PU.1 following induction by TGFβ. Evidence is rapidly accumulating that IL-9 is the mediator in acute contact dermatitis, asthma, inflammatory bowel disease, pediatric atopy, and giant cell arteritis.
In addition to ensuring the self-renewal of the hematopoietic stem cell (HSC), PU.1 governs cell fate determination in a dosage- and cell-stage dependent fashion. Elevated PU.1 activity is required to drive differentiation of the HSC towards the myeloid lineages (the common myeloid progenitor), at which point continued PU.1 activity induces the terminal development of macrophages and granulocytes, while a tapering of PU.1 activity leads to erythrocytes. At lower concentrations, PU.1 also drives the initial differentiation of the HSC to the common lymphoid progenitor, at which stage a switch in PU.1 dosage induces terminal differentiation into B-(high PU.1) or T-lymphocytes (low PU.1). Since hematopoietic cell fate decisions require multiple transcription factors, often acting in antagonistic fashion [e.g., ↓ PU.1/↑ Ets-1 during T cell development); ↓ PU.1/↑ C/EBPα in regulation of macrophage and neutrophils], PU.1 inhibitors are expected to be useful in a cocktail of other transcriptional modulators, to induce the differentiation of appropriate progenitors into desired cell types. The expected usefulness of PU.1 inhibitors as a cell-reprogramming agent is highlighted by a reversal in PU.1/Ets-1 antagonism in the specialization of mature T cells into subtypes such as Th9.
Typical of ETS-family transcription factors, DNA site recognition by PU.1 requires contact with the major groove, at consensus sites harboring the 5′-GGAA/T-3′ sequence specific for the ETS family. Additional contacts with the adjacent DNA minor groove confer selectivity for certain ETS paralogs, such as AT-rich sequences for PU.1. PU.1 inhibitors targeting DNA in the minor groove, by targeting the AT-rich sequences, therefore lead to inhibition of PU.1 binding in the major groove via an allosteric mechanism.
There is a need for novel compounds with enhanced inhibitory potency against PU.1. There is a need for improved methods for treating cancers, including hematologic cancers such as leukemia, as well as other conditions associated with PU.1 dysfunction. The present invention addresses these needs.